Distributions of high-density lipoprotein particle components
in human follicular fluid and sera and their associations with
embryo morphology parameters during IVF
R.W. Browne1,4, W.B. Shelly2, M.S. Bloom3, A.J. Ocque1, J.R. Sandler2, H.G. Huddleston2
and V.Y. Fujimoto2
1Department of Biotechnical and Clinical Laboratory Sciences, University at Buffalo, State University of New York, 26 Cary Hall, 3435
Main Street, Buffalo, NY 14214, USA;2Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, at
San Francisco, San Francisco, CA, USA;3Department of Environmental Health Sciences, University at Albany, State University of
New York, Rensselaer, NY, USA
4Correspondence address. Fax: þ1-716-829-3601; E-mail: firstname.lastname@example.org
BACKGROUND: High-density lipoprotein(HDL) is the sole lipoproteinpresent in follicular fluid (FF).The objectives
of this study were to examine HDL lipid composition and associated enzyme activities in FF and serum and to relate
these levels to embryo morphology parameters in women undergoing in vitro fertilization (IVF). METHODS: Serum
and FF were prospectively obtained from 60 women undergoing IVF. HDL lipids, apolipoprotein AI (ApoAI), para-
oxonase 1 (PON1) and paraoxonase 3 (PON3) activities were determined. Bivariate analysis and ordinal logistic
regression models were employed to examine the associations between biochemical measures and embryo morphology
parameters [embryo cell number (ECN) and embryo fragmentation score (EFS)] as surrogate markers of oocyte
health. RESULTS: All biochemical parameters were significantly (P < 0.05) lower in FF than serum except PON3
levels which were significantly higher. FF-HDL cholesterol (OR 0.66, 95%CI 0.46–0.96) and ApoAI (OR 0.13,
95%CI 0.03–0.97) levels were negative predictors for EFS; however, their effects were not independent and the
level of one moderated the effect of the other. Limited to Day 3 embryo transfers, FF-PON1-arylesterase activity
was a significant positive predictor for ECN (OR 1.09, 95%CI 1.01–1.17). CONCLUSIONS: In this pilot study,
our data suggests that HDL and its component proteins within FF may play protective roles in the health of the
human oocyte and subsequent early embryo development. We describe for the first time the activities of PON1 and
PON3 in FF. We suspect that PON3 activity may be locally generated due to higher activities in FF compared with
Keywords: high-density lipoprotein; follicular fluid; embryo cytoplasmic fragmentation; in vitro fertilization; paraoxonase
HDL-cholesterol has been classically recognized as the mol-
ecule responsible for providing cholesterol as the substrate
for de novo steroidogenesis within the ovarian follicle
(Simpson et al., 1980; Jaspard et al., 1996, 1997; Azhar
et al., 1998; Li et al., 2001). Yet, high-density lipoprotein
(HDL) also exhibits potent anti-inflammatory and antioxidant
properties that contribute to its general antiatherogenic and
cytoprotective effects (Ansell et al., 2003; Negre-Salvayre
et al., 2006). Determinants of HDLs antioxidant/anti-
inflammatory activities include its size and density (Nobecourt
et al., 2005), lipid composition (McPherson et al., 2007), the
apolipoprotein moiety (ApoAI) and the presence of associated
enzymes including paraoxonase 1 and 3 (PON1 and PON3)
(von Eckardstein et al., 2005; Negre-Salvayre et al., 2006).
HDL is the sole lipoprotein present in follicular fluid (FF)
due to the porosity of the follicle membrane which is per-
meable to serum proteins up to 300 kDa in size thus excluding
low-density lipoprotein (LDL), very low-density lipoprotein
(VLDL) and larger HDL2s which are not expected to filter
through the follicular basement membrane (Le Goff, 1994;
Jaspard et al., 1996). HDL particles consist of a lipid mono-
layer and core in addition to protein components including apo-
lipoproteins (ApoAI, AII and AIV), and lipolytic and
antioxidant enzymes. Among these proteins, ApoAI and
PONs appear to have the most dominant antioxidant activities
(Wu et al., 2007).
The PON gene family in humans has three members, PON1,
PON2 and PON3 (Primo-Parmo et al., 1996). PON1 is syn-
thesized in the liver and carried in the blood associated
# The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
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Human Reproduction Vol.23, No.8 pp. 1884–1894, 2008
Advance Access publication on May 16, 2008
exclusively with HDL where it is the major determinant of
HDLs ability to protect LDL (Mackness et al., 1991), cells
(Draganov et al., 2000) and lipids from oxidative and peroxida-
tive modification (Draganov et al., 2005; Teiber et al., 2007).
Recently, PON1 protein has been identified in FF by proteomic
analysis (Angelucci et al., 2006). PON1 activity is currently
characterized using two substrates, paraoxon (PON activity)
and/or phenyl acetate (arylesterase activity); however, these
activities have not been described in FF. When PON3 protein
was first discovered, it was localized to HDL and demonstrated
to have similar antioxidant properties to PON1 despite the fact
that it is ?100 times lower in concentration in serum than
PON1 (Reddy et al., 2001; Draganov, 2007). The presence of
PON3 in FF has, to our knowledge, not been previously
In female reproduction, evidence is accumulating on the role
of reactive oxygen species, with oxidative stress (OS) in the
Graafian follicle proving to be an important process in defining
female reproductive potential via oocyte genetic and cyto-
plasmic integrity (Agarwal et al., 2003). Specific antioxidant
enzymes and radical scavengers are present in FF (Bisseling
et al., 1997; Sabatini et al., 1999; Pasqualotto et al., 2004) as
are several markers of oxidative damage (Wiener-Megnazi
et al., 2004; Das et al., 2006); however, the effects of these
markers on oocyte quality, embryo quality and other clinical
reproductive outcomes have thus far produced conflicting
Given the unique properties of HDL and its component pro-
teins together with their isolated presence in FF in the absence
of LDL, we sought to quantify the lipid and apolipoprotein
composition of FF-HDL relative to serum obtained from
patients undergoing in vitro fertilization (IVF) and examine
possible associations with morphologic indices of embryo
quality. Furthermore, the novel quantification and characteriz-
ation of PON1 and PON3 activities in FF are described in the
context of this prospective pilot study.
Materials and Methods
There were 60 patients undergoing IVF treatment at the UCSF Center
for Reproductive Health recruited into this study. Subjects were
enrolled prospectively prior to completion of their IVF cycle with
full informed consent. No participants actively withdrew from the
study after consent. The study protocol and consenting process were
approved by the UCSF Committee on Human Research. Participants
underwent gonadotrophin-induced ovarian stimulation per clinic pro-
tocols: (i) down-regulated luteal leuprolide acetate; (ii) microdose leu-
prolide acetate flare; (iii) gonadotrophin-releasing hormone (GnRH)
antagonist with/without estrogen priming and (iv) demi-halt (luteal
leuprolide acetate discontinued prior to gonadotrophin initiation).
Transvaginal ultrasonography and serum estradiol levels were per-
formed routinely during ovarian stimulation to assess ovarian follicle
maturation. When a sufficient number of mature-sized follicles
(?16 mm diameter) developed, human chorionic gonadotrophin
(5000–10 000 IU) was administered subcutaneously 36 h prior to
scheduled oocyte retrieval. A blood sample was obtained from the
patient 15–30 min prior to the oocyte retrieval procedure via an intra-
venous line placed for anesthesia and hydration purposes. FF from an
individual mature 18-20 mm follicle was aspirated using a single
lumen 17 gage 35 cm aspiration needle guided by transvaginal ultra-
sonography. After identification and removal of the cumulus mass,
the FF along with serum was processed immediately. The processed
samples were frozen at 2808C and stored until shipment to the OS
Laboratory at the University of Buffalo. A total of 60 individually
aspirated follicles from 60 patients were included in this study.
Intracytoplasmic sperm injection (ICSI) was performed in most
cases based on abnormal sperm parameters from prior semen analyses.
Otherwise, conventional insemination was performed. For ICSI cases,
oocytes were denuded of cumulus granulosa cells prior to sperm injec-
ified for all ICSI cases. All metaphase-II oocytes were fertilized, with
sperm collected from the male partner on the day of oocyte collection
or a frozen sperm specimen from the male partner or sperm donor,
standard commercially-available culture media (Vitrolife Series
G1.3w, Kungsbacka, Sweden) for 24–48 h prior to embryo mor-
phology evaluation. Embryo morphologic indices were determined
using an inverted phase contrast Nikon Diaphotwmicroscope, and
included individual embryo cell number (ECN) and individual
embryo fragmentation score (EFS), each assessed on the day of
licles. Embryo morphology assessments for all study participants were
madeonly byexperienced embryologistswho were blindedto any bio-
in our laboratory undergo routine quality control assessment to mini-
mize inter-observer variability. Similarly, the OS laboratory was
blinded to any clinical outcomes data during assay analyses.
Embryo fragmentation scoring was based on the degree of fragmen-
tation present per embryo assessed on the day of embryo transfer with
the following criteria: Grade 1, 0% fragmentation; Grade 2, 1–10%
fragmentation; Grade 3, 11–25% fragmentation; Grade 4, 26–50%
fragmentation; and Grade 5, 51% or greater fragmentation. ECN
and degree of fragmentation are both considered important clinical
markers of embryo quality and viability (Puissant et al., 1987; Steer
et al., 1992).
HDL fractionation and lipid composition analysis
Serum and FF-HDL fractions were prepared by selective precipitation
methods (Gidez et al., 1982), since ultracentrifugation is known to
disrupt lipoprotein structure (Kunitake and Kane, 1982), and it has
been suggested that HDL components may be redistributed among
lipoproteins during ultracentrifugation (Cabana et al., 2003). Despite
the absence of non-HDL lipoproteins in FF, the precipitant was
added to create the same condition as in serum. Briefly, 0.1 vol of
heparin-MnCl2(40 000 USP units/ml heparin in 1.0 mol/l MnCl2)
solution was added to 1.0 vol of sample. After 5 min incubation at
room temperature, the samples were centrifuged at 16 000 g in a
microcentrifuge, and the supernatant was collected.
Total lipid analysis
ApoAI, cholesterol, phospholipids and triglycerides levels were
measured using diagnostic reagent kits from Wako Diagnostics Inc.
(Richmond, VA, USA) adapted to the Cobas Fara II (Hoffmann–La
Roche & Co., Switzerland) automated chemistry analyzer. The analy-
sis parameters of these assays were modified by increasing the sample
size by a factor of 3–5 times to account for the lower lipid concen-
trations encountered in the isolated HDL preparations. All analyses
were performed in duplicate and reported in mg/dl. The analytical
coefficient of variation of each assay was ,5%.
HDL composition and embryo morphology parameters
PON1 arylesterase and PON activities
The arylesterase and PON activities of PON1 were determined exactly
as recently described (Browne et al., 2007). Diethyl p-nitrophenyl
phosphate (paraoxon), 98.0%, was obtained from Chem Service
(Westchester, PA, USA). PON activity was determined by the rate
of formation of p-nitrophenol at 412 nm using 1 mmol/l paraoxon
as the substrate in 50 mmol/l glycine buffer, pH 10.5, with
1.0 mmol/l CaCl2. The activity was expressed as IU/l based on the
molar absorptivity (18 290) of p-nitrophenol at 405 nm, at pH 10.5.
Arylesterase activity was determined by the rate of formation of
phenol at 270 nm using 4 mmol/l phenyl acetate as the substrate in
20 mM Tris–HCl, pH 8.0, with 1.0 mM CaCl2. The activity,
expressed as kU/l, was based on the molar absorptivity (188) of
phenol at 285 nm, at pH 8.0. For all assays, water blanks were used
to correct for non-enzymatic hydrolysis.
PON3 is the only member of the PON family which metabolizes bulky
drug substrates such as lovastatin and simvastatin (Draganov et al.,
2005). We have instituted and validated a method recently described
by Suchocka et al. (2006). PON3 activity was determined by
RP-HPLC monitoring of the conversion of simvastatin (SV) to
b,d-dihydroxyacid simvastatin (SVA) during incubation with serum
or FF. The basal reaction mixture [940 ml of 20 mmol/l Tris–HCl
(pH 7.6) containing 0.9 mmol/l CaCl2and 40 mmol/l of physostyg-
mine] was pre-incubated for 10 min at 378C in water-bath and then
10 ml of SV (0.5 mg/ml in methanol) was added. The reaction was
initiated by adding 50 ml of serum or FF and the enzymatic reaction
was stopped after 1 h by deproteinization with 1.0 ml cold acetonitrile.
HPLC analysis of SV and SVA was carried out using a Shimadzu
SPD-10AV diode array and Sil10A autoinjector (Shimadzu Scientific
Instruments, Inc., Columbia, MD, USA). SV and SVA separations
were performed on a SupelcoSil C18, 5 mm, 250 mm ? 4.60 mm
column, at room temperature 22–258C. Peak detection was performed
at l ¼ 239 nm. The PON3 activity in serum or FF was expressed as
the rate of formation of SVA in pmol/min/ml.
ofa LC-10AD Vppump,
Enzyme inhibition studies
To confirm that the enzymatic activities measured were due to PON
enzymes and not other hydrolytic enzymes, we performed several
inhibition experiments. PONs are calcium-dependent enzymes and
therefore inhibited by EDTA treatment. Inhibition by EDTA was per-
formed by diluting samples 1:1 in buffer (50 mM Tris–HCl,
40 mmol/l EDTA, pH 7.4) and incubating for 16 h at room tempera-
ture. Buffer for the controls contained 1 mM CaCl2instead of EDTA.
To rule out the possibility of cholinesterase activity in our assays,
samples were inhibited with physostigmine (a cholinesterase inhibi-
tor), phenylmethanesulfonyl fluorine (PMSF; a serine protease inhibi-
tor) and tetraisopropyl pyrophosphoramide (iso-OMPA; a selective
butrylcholinesterase inhibitor). Each inhibition was performed by
adding 2 ml of the inhibitor (200 mmol/l in methanol), or methanol
for controls, to samples that had been diluted 1:2 in buffer
(50 mmol/l Tris–HCl, 1 mmol/l CaCl2, pH 7.4) so that the final con-
centration of inhibitor was 2 mmol/l. Samples were then incubated at
room temperature for 2 h. Aliquots of the inhibited and control
samples were analyzed as described above.
All statistical analyses were performed using SAS version 9.1.3 with
statistical significance defined as P , 0.05 for a two-tailed test. Non-
parametric analyseswere favoredincludingSpearman rank
correlation coefficients (rSp) for continuous and ordinal data;
Wilcoxon rank sum tests were used for paired differences, and
Mann–Whitney U-test, x2-test or Fisher’s exact tests, as appropriate,
were used for categorical data. Differences between the median values
for sera and FF concentrations of each biochemical analyte were
assessed as were correlations between serum and FF concentrations
and with demographic factors.
Correlations were evaluated between concentrations for bio-
chemical analytes and embryo morphology parameters among
oocytes retrieved from the 60 sampled follicles (i.e. one per
woman). These correlations, stratified by body compartment (i.e.
serum or FF) were employed to select potential predictors using
multivariable ordinal logistic regression, also known as pro-
portional odds, models (McCullagh, 1980). This extension of
binary logistic regression, assuming proportionality, estimates the
log odds of a subject’s outcome falling into the kth (k ¼ 1, 2 and
3) outcome category or lower across k21 ordered response
variate categories. A more efficient capture of the ordered infor-
mation inherent in an ordinal response variate, and consequently
greater power and precision, may be effected than that offered by
dichotomization and binary logistic regression. Biochemical corre-
lates of embryo morphology parameters, with P , 0.15, were
entered into a forward stepwise selection procedure (entry/exit cri-
teria 0.05/0.10) as potential predictors for EFS (1/2/3–5; n ¼ 8/
13/9) and for ECN (2–4 cells/5–7 cells/8–11 cells; n ¼ 2/7/
13), with the latter limited to Day 3 embryo transfers. ‘Unselected’
potential biochemical predictors, age, BMI and patient race/ethni-
city were considered as potential confounders for ‘selected’
models. Confounding was defined as a minimum 15% change in
the magnitude for biochemical predictors following entry of the
covariate into the ‘selected’ model (Kleinbaum et al., 1998). Inter-
action terms were considered between ‘selected’ predictors and any
confounders retained in the models, these were included in final
models where P , 0.10. Multiple imputation, employing the EM
algorithm, was used to impute missing values (a consequence of
limited FF sample volume) during ordinal logistic regression mod-
eling (Horton and Kleinman, 2007).
The study sample
The complete demographic, treatment and treatment outcome
covariates for the study population are shown in Table I. A
large range for age (29–44 years) and BMI (18–34 kg/m2)
were represented among the 60 female study participants.
The relatively high proportion of Asian participants (36.7%)
reflects the population of the San Francisco Bay area from
which the study participants were recruited. Approximately
40% of participants had undergone prior IVF procedures.
Male factor (42%) was the most prevalent infertility diagnosis
among study participants, followed by unexplained (22%), and
diminished ovarian reserve (15%). Approximately two-thirds
of study participants received a down-regulated luteal lupron
stimulation protocol. ICSI was employed for 78% (n ¼ 47)
PON activity validation
to PONs, we performed inhibition studies in lieu of having
specific PON1 or PON3 inhibitors which have yet to be ident-
ified. To conduct these inhibition studies, three FF samples
Browne et al.
were pooled, divided and then treated with inhibitors as
described in the Methods and Materials. The results are shown
vastatinase and PON1 arylesterase activities were inhibited due
cating that the majority of the reported activities cannot be
attributed to the activities of serine proteases, cholinesterases
or butrylcholinesterases, respectively. Nearly identical results
were obtained for PON1 PON activities (data not shown).
Characterization of serum and FF-HDL levels and PON
Box and whisker plots describing the distributions for levels of
HDL lipids, PON1 and PON3 enzyme activities, in serum and
FF, are presented in Fig. 2. Median FF-HDL component con-
centrations were lower for cholesterol (20.9 versus 37.2, P ,
0.0001, n ¼ 59), phospholipids (73.1 versus 94.6, P ,
0.0001, n ¼ 40) and ApoAI (105.5 versus 157.3, P ¼ 0.001,
n ¼ 59) when compared with serum HDL, whereas triglycer-
ides levels were not different (7.5 versus 8.4, P ¼ 0.546, n ¼
39). Except for triglycerides (rSp¼ 0.09, P ¼ 0.597), levels
of HDL lipids were positively correlated between FF and
serum (rSp¼ 0.44–0.95, P ? 0.001). Median sera activities
were also greater than FF for PON1-arylesterase (154.2
versus 93.2, P , 0.0001, n ¼ 60) and PON1-PON (186.5
versus 116.4, P , 0.0001, n ¼ 60), and values were highly cor-
related between these compartments (rSp¼ 0.74–0.95, P ,
0.0001). In contrast, median PON3-simvastatinase activity
was lower in serum than in FF (10.6 versus 16.5, P ¼ 0.006,
n ¼ 58) with an attenuated, yet statistically significant, corre-
lation between the two compartments (rSp¼ 0.36, P ¼
0.006). With the exception of several observations for
HDL-triglycerides (n ¼ 23, 41.0%) and PON3 (n ¼ 2, 3.4%),
the observed pattern of differences between serum and FF
was consistent for all study subjects.
The correlations between serum and FF-HDL components
levels suggest that FF-HDL is blood-derived; however, it is sig-
nificant to note that FF-HDL particle composition is propor-
tionally different to that in serum. ApoAI is a surrogate
marker of the number of HDL particles. Previous studies indi-
cate that FF-HDL particle ApoAI is not different from blood
plasma and can be useful for relative composition comparisons
(Jaspard et al., 1997). Upon normalization of HDL components
per molecule of ApoAI (i.e. HDL component concentrations
divided by ApoAI concentration), it was evident that
FF-HDL is 25% lower in cholesterol content (P , 0.001),
10% higher in phospholipids content (P , 0.001) and 40%
higher in triglyceride content (P , 0.01) when compared
with serum by the Wilcoxon rank sum test. These ApoAI-
normalized values are reported here to be descriptive and high-
light the different HDL particle composition in FF. These nor-
malized values were not used during subsequent ordinal
logistic regression analyses with clinical outcomes as measure-
ment error and bias may be introduced as previously demon-
strated for lipid normalization (Schisterman et al., 2005).
Substantial intercorrelations were measured among the lipid
components of HDL and the levels of PON1 activities in FF, as
in Table II. HDL-cholesterol, HDL phospholipids and ApoAI
all correlated significantly with each other (rSp¼ 0.36–0.62,
Table I. Characteristics of study participants by demographic, treatment and
treatment outcomes covariates (n ¼ 60).
Demographic covariatesMean (SD)Median Range
Ethnicity, n (%)
Number of prior IVF cyclesb
Diagnosis, n (%)c
Protocol, n (%)
1.9 (1.5)1.0 1.0–7.0
BMI, body mass index; DOR, diminished ovarian reserve; E2, estradiol; IVF,
in vitro fertilization; SAB, spontaneous abortion.
an ¼ 3 missing values.
bn ¼ 24 (40.0%) patients with a prior history of IVF treatment.
cDiagnoses are mutually exclusive, n ¼ 1 missing value.
Figure 1: Inhibition of PON1 arylesterase activity and PON3 simvas-
tatinase activity in serum (white bars) and follicular fluid (black bars)
by EDTA and three enzyme inhibitors. Results are expressed as a
percentage of the uninhibited, control activity (kIU/l for PON1 and
pmol/min/ml for PON3).
HDL composition and embryo morphology parameters
P ? 0.022), probably due totheir common location on the HDL
particle. PON1 arylesterase activity also correlated with these
parameters albeit to a lesser degree (rSp¼ 0.23–0.36), reach-
ing significance only in association with ApoAI (P ¼ 0.005).
Interestingly, a negative association was suggested between
(r ¼ 20.23, P ¼ 0.087), although this correlation did not
reach statistical significance. A similar pattern of correlations
was measured among serum analytes (data not shown).
Correlation of HDL and PON activities with embryo
Metaphase-II oocytes were harvested from ?83% (n ¼ 50) of
individually sampled follicles. Approximately 64% (n ¼ 32) of
these oocytes were normally fertilized as indicated by 2PN
embryos. Significant negative correlations to EFS were
measured for FF-HDL cholesterol (rSp¼ 20.59, P ¼ 0.001),
FF ApoAI (rSp¼ 20.54, P ¼ 0.002), serum-HDL cholesterol
(rSp¼ 20.53, P ¼ 0.003), serum ApoAI (rSp¼ 20.50, P ¼
0.005), aswell asserum
(rSp¼ 20.39, P ¼ 0.032) (Table III). Adjusting for day of
embryo transfer, which was significantly associated with
ECN [2-cells versus 8-cells (P , 0.0001) for Day 2 versus
Day 3, respectively], no statistically significant correlations
were measured between ECN and any FF or serum analytes.
There was a positive correlation between FF-PON1-arylester-
ase activity and ECN, but this did not reach statistical signifi-
cance (rSp¼ 0.29, P ¼ 0.122). There were no statistically
Figure 2: Box and Whisker plots describing the distributions for lipid components of fractionated HDL (A) and enzyme activities (B) from fol-
licular fluid and serum.
HDL-Ch, high-density lipoprotein cholesterol; HDL-Pl, high-density lipoprotein phospholipids; HDL-Tr, high-density lipoprotein triglycerides;
PON1-Ary, paraoxonase 1 arylesterase activity; PON1-Par, paraoxonase 1 paraoxonase activity; PON3-SIM, paraoxonase 3 simvastatinase
Browne et al.
significant correlations between embryo morphology par-
ameters and BMI or patient race/ethnicity, but a correlation
of ‘borderline’ significance was measured for age and ECN
(rSp¼ 20.31, P ¼ 0.088).
Ordinal logistic regression models for embryo morphology
FF ApoAI (OR 0.66, 95%CI 0.46–0.96) and HDL-cholesterol
Figure 2: Continued
Table II. Spearman rank correlation coefficients (P-values) among biochemical analytes measured in ‘follicular fluid’ specimens.
HDL-ChHDL-Pl HDL-TrApoAI PON1-Ary PON1-Par PON3-Sim
HDL-Ch (n ¼ 60)
HDL-Pl (n ¼ 40)
HDL-Tr (n ¼ 39)
ApoAI (n ¼ 60)
PON1-Ary (n ¼ 60)
PON1-Par (n ¼ 60)
PON3-Sim (n ¼ 60)
Correlations with P , 0.05 in bold typeface. ApoAI, apolipoprotein AI; HDL-Ch, high-density lipoprotein cholesterol; HDL-Pl, high-density lipoprotein
phospholipids; HDL-Tr, high-density lipoprotein triglycerides; PON1-Ary, paraoxonase 1 arylesterase activity; PON1-Par, paraoxonase 1 paraoxonase activity;
PON3-SIM, paraoxonase 3 simvastatinase activity.
HDL composition and embryo morphology parameters
for EFS (Table IV). Furthermore, a positive interaction term
retained in this model (OR 1.02, 95%CI 1.00–1.03) suggested
that the effects of FF ApoAI and HDL-cholesterol were to
some extent interdependent. For example, an increase in either
FF-HDL cholesterol, or in FF ApoAI, elicited a slight reduction
in the ‘protective’ effect of the other on EFS. Using serum,
resultsweresimilarto theFFmodelsforEFS;however,no inter-
action was retained in the model, and effect estimates for ApoAI
(OR 0.95, 95%CI 0.91–1.00) and HDL-cholesterol (OR 0.95,
95%CI 0.88–1.03) were substantially attenuated. In the model
for ECN, limited to Day 3 embryo transfers (n ¼ 22),
PON1-arylesterase activity in FF was a significant positive pre-
dictor (OR 1.09, 95%CI 1.01–1.17). No ordinal logistic
regression model was generated for serum measures and ECN.
The current study was intended to generate hypotheses regard-
ing potential clinical markers of oocyte quality, employing
embryo morphology parameters, for consideration in a future
investigation utilizing a larger sample size. The fundamental
importance of this study is the identification of clinical
markers that correlate with human embryo morphology par-
ameters in vitro. Furthermore, this is the first quantification
and characterization of PON1 and PON3 enzyme activities in
human ovarian FF. Whereas other studies have focused on
the composition of FF-HDL with respect to de novo steroido-
genesis, HDL and its component lipids (cholesterol, phospho-
lipids and triglycerides) and proteins (ApoAI, PON1 and
PON3) have, to the best of our knowledge, never been evalu-
ated with respect to their effects on embryo quality during IVF.
Within the scope of this preliminary work, we have noted
several interesting findings with regard to HDL composition
of FF and IVF embryo morphology parameters. First,
HDL-cholesterol and ApoAI concentrations in both FF and
serum, both indicators of HDL particle concentration, were
negatively associated with Day 3 EFS. However, these
effects were likely interdependent as suggested by the inclusion
of a statistical interaction in the relevant ordinal logistic
regression model. Second, FF-PON1-arylesterase activity was
a positive predictor of Day 3 ECN. Finally, although PON3
had no correlation with embryo morphology parameters, its
higher activity in FF relative to serum suggests that it may be
locally generated within the follicle.
The negative association between embryo fragmentation
and implantation rates with IVF has been firmly established
(Giorgetti et al., 1995; Alikani et al., 1999; Ebner et al.,
2001; Volpes et al., 2004; Holte et al., 2007). Others have
described an association between embryo fragmentation and
reduced blastocyst formation (Rijnders and Jansen, 1998;
Hardy et al., 2003; Stone et al., 2005; della Ragione et al.,
2007). The positive association between embryo fragmentation
and programmed embryonic cell death was initially proposed
by Jurisicova et al. (1996). Subsequent studies have confirmed
this association with additional evidence for an OS-mediated
etiology (Yang et al., 1998; Bedaiwy et al., 2004, 2006).
Bencomo et al. (2006) described an association between gran-
ulosa cell apoptosis and embryo fragmentation. However,
others have called into question the relationship between
embryo fragmentation and cellular apoptosis (Van Blerkom
et al., 2001, 2002; Alikani et al., 2005). Our preliminary find-
ings support the hypothesis that HDL, with its component pro-
teins including PON1, may have an important cytoprotective
Table IV. Ordinal logistic regression models for embryo quality indicators among oocytes harvested from sampled follicles.
Outcomen Compartment Predictor
b (P-value)OR 95% CI OR
Coefficients with P , 0.05 in bold typeface. ApoAI, apolipoprotein AI; ECN, embryo cell number; EFS, embryo fragmentation score; FF, follicular fluid;
HDL-Ch, high-density lipoprotein cholesterol; PON1-Ary, paraoxonase 1 arylesterase activity.
aInteraction term applies to FF ApoAI and FF-HDL-cholesterol interaction. Higher EFS is equivalent to greater embryo cytoplasmic fragmentation.
bLimited to embryos transferred on Day 3 following fertilization.
Table III. Spearman rank correlation coefficients (P-values) between
biochemical analytes and embryo quality indicators.
EFS (n ¼ 30)ECN (n ¼ 31)a
20.23 (0.319)n ¼ 21
0.04 (0.862)n ¼ 20
0.20 (0.308)n ¼ 29
0.14 (0.556)n ¼ 21
20.20 (0.408)n ¼ 20
20.11 (0.585)n ¼ 30
20.22 (0.336)n ¼ 21
20.01 (0.977)n ¼ 21
20.23 (0.337)n ¼ 21
20.12 (0.621)n ¼ 21
Sample sizes vary for certain analytes due to limited follicular fluid sample
volume; Correlations with P , 0.05 in bold typeface. ApoAI, apolipoprotein
AI; HDL-Ch, high-density lipoprotein cholesterol; HDL-Pl, high-density
lipoprotein phospholipids; HDL-Tr, high-density lipoprotein triglycerides;
PON1-Aryl, paraoxonase 1 arylesterase activity; PON1-Par, paraoxonase1
paraoxonase activity; PON3-SIM, paraoxonase 3 simvastatinase activity.
aAdjusted for day of embryo transfer.
Browne et al.
effect on the oocyte and its surrounding granulosa cells via
mechanisms that are currently unknown.
The importance of HDL to oocyte potential is exemplified by
studies in SR-BI (scavenger receptor type B class I) knockout
mice (Trigatti et al., 1999; Miettinen et al., 2001; Yesilaltay
et al., 2006). SR-BI mediates cellular uptake of HDL by
binding HDL via its apolipoprotein components and regulating
the intracellular movement of cholesterol. SR-BI knockout
female mice were identified to be infertile as a result of
embryos that uniformly arrested in early stages of develop-
ment, primarily at the 1- and 2-cell stages (Trigatti et al.,
1999). The SR-BI knockout model displays a distinctive lipo-
protein abnormality characterized by abnormally-large HDL
particles with a high unesterified cholesterol:total cholesterol
ratio (UC:TC ratio). Restoration of fertility occurs when
SR-BI2/2ovaries are transplanted into wild-type mice indicat-
ing that the impairment is not due to the SR-BI receptor
deficient ovary, but rather the abnormal HDL particle produced
by the SR-BI knockout murine liver (Miettinen et al., 2001).
Furthermore, treatments of SR-BI2/2mice with probucol (to
correct the UC:TC ratio) restores fertility (Miettinen et al.,
2001), whereas partial restoration of fertility via inactivation
of the ApoAI gene and reduction of total cholesterol do not nor-
malize HDL particle size (Yesilaltay et al., 2006). These
studies suggest the importance of HDL concentration, structure
and composition in determining mammalian female reproduc-
Determination of the precise mechanism by which HDL may
influence reproductive potential will require further studies;
however, it is tempting to speculate. Potential mechanisms
might include the protection against OS by the potent antioxi-
dant composition of HDL, the regulation of steroidogenesis
since HDL is the primary source of cholesterol within the fol-
licle, or impairment of transport processes whereby HDL is
unable to deliver, or remove by efflux, key compounds to/
from the ovary. Alternatively, the role of HDL may be signifi-
cantly related to the ovarian activity of lipolytic enzymes. Our
analyses of HDL components indicate not only that the absol-
ute levels of HDL and its components differ between FF and
blood serum but also that the lipid composition of FF-HDL par-
ticles is proportionally cholesterol-poor and phospholipids-rich
compared with serum-HDL particles. These results are consist-
ent with previous findings that FF-HDL contains a higher pro-
portion of smaller HDL3particles relative to larger HDL2
particles (Le Goff, 1994; Jaspard et al., 1996). HDL2s are not
expected to filter through the follicular membrane and their
presence in FF is thought to originate from the remodeling of
smaller HDL3 particles involving phospholipids transfer
protein (PLTP) and hepatic triacylglycerol lipase. It has been
shown that PLTP can convert HDL3particles into two popu-
lations of large (HDL2) and small particles (pre-bHDL) (Tu
et al., 1993) and mRNAs of PLTP have been detected in
large amounts in human ovaries (Albers et al., 1995).
Hepatic triacylglycerol lipase has been detected in ovaries
and adrenal glands, with specific activity in FF (Hixenbaugh
et al., 1989). All of these findings point to the significant remo-
deling of HDL in FF with a high proportion of HDL3particles.
Furthermore, ApoAI has become increasingly important in the
maintenance of a functional HDL particle with antiatherogenic
properties (Wu et al., 2007). Thus, it is not surprising that our
data would support the concept that ApoAI acts in concert with
HDL-cholesterol within the HDL particle to influence embryo
In addition, we have demonstrated the presence of both
PON1 and PON3 activity in human FF which, to our knowl-
edge, has never been reported before in the literature. On the
basis of the patterns of inhibition generated using several
experimental conditions, it is unlikely that these activities
can be attributed to enzymes other than PONs (Teiber et al.,
2007). The data suggest an association between PON1 activity
and HDL within the FF which is expected given the exclusive
association of PON1 with HDL in the blood (Eckerson et al.,
1983; Blatter Garin et al., 2006). This association argues for
passive transfer of PON1 together with HDL through capillary
mechanisms from blood into the FF compartment during folli-
culogenesis. Our observation that PON1-arylesterase activity
was positively association with ECN, and consequently
embryo cleavage, is rational given the established antioxidant
HDL-cholesterol, ApoAI and PON1 activity with embryo mor-
phologic parameters suggest that the overall HDL status of the
follicle is important to oocyte health.
Unlike PON1, PON3 enzymatic activity is higher in FF rela-
tive to serum and we did not observe an association between
PON3 and HDL-cholesterol or ApoAI. Thus, PON3 activity
appears to be independent from HDL, possibly existing in FF
in a non-HDL-associated state. In mammals, PON1 is syn-
thesized in the liver and carried exclusively in the circulation
bound to HDL (Eckerson et al., 1983). In contrast, PON3
appears to be produced by multiple tissues, including liver,
human gastrointestinal tract, mouse airway epithelium, macro-
phages, kidney, lung, brain and adipose tissues (Shih et al.,
2007). Our observations suggest that the increased PON3
enzyme activity in FF may be due to de novo production in
the human follicle with possible origination from granulosa
An acknowledged limitation of this study is the small sample
size. However, the number of associations found, despite this
limitation, point to a potentially important role for HDL,
ApoAI and PON1 activities in the overall health of the devel-
oping follicle and its resulting oocyte and embryo. Another
limitation of this study involves the potential influence of
sperm on early embryo development. While current literature
suggests an important role for the oocyte in the etiology of
early embryo cytoplasmic fragmentation, based on oocyte mor-
phology and shared sibling oocyte studies (Serhal et al., 1997;
Xia, 1997; Loutradis et al., 1999; Ebner et al., 2000; Salumets
et al., 2002), a role for sperm in early embryo development has
also been described (Tesarik et al., 2002, 2004; Tomsu et al.,
2002; Seli and Sakkas, 2005). Tomsu et al. (2002) described
a correlation between sperm mean head density and embryo
quality that included both blastomere symmetry and degree
of fragmentation in an IVF patient population. Tesarik et al.
embryos generated from the same sperm source despite differ-
ent oocyte sources. However, using shared sibling oocytes,
HDL composition and embryo morphology parameters
Tesarik et al. (2004) found no effect of sperm DNA fragmenta-
tion using the TUNEL assay on early zygote morphology.
Neither of the Tesarik et al. studies used embryo fragmentation
during the cleavage stage as an outcome variable. Salumets
et al. (2002) utilized a shared sibling oocyte model to demon-
strate that embryo fragmentation using standard morphologic
criteria similar to ours was strongly correlated with the
oocyte source in 59 ovum donation cycles. This study effec-
tively articulated the effect of intrinsic oocyte quality on
embryo fragmentation in the cleavage stage embryo. This
apparent dependence of early embryo morphologic obser-
vations at least in part on oocyte integrity provided the basis
for using embryo morphology parameters as outcomes of inter-
est in our study.
In summary, HDL metabolism within the follicle is
undoubtedly complex, influenced by HDL composition and
size, follicular vascularity, varying degrees of follicular
HDL particle remodeling and variable utilization of HDL
and its components by follicular cells. Within the framework
of this complexity, our study points to HDL playing a poten-
tial role in the health of the human oocyte with embryo
fragmentation and embryo cell number serving as surrogate
markers of oocyte health. Our pilot study establishes the
possibility that HDL and/or its component proteins have the
potential to predict embryo fragmentation and cell number
in women undergoing IVF. If validated in embryo cohort
data employing a larger sample size, these biomarkers may
prove to be clinically relevant for predicting patients more
likely to have embryo fragmentation and cleavage problems.
Except for the PON3 simvastatinase activity, the assays
employed here have been adapted from commercially avail-
able reagent kits. The lipid and lipoprotein analyses are routi-
nely available in most clinical laboratories and could be
rapidly analyzed to provide timely information prior to or
during the conduct of IVF treatment.
Further efforts are currently underway to examine the varia-
bility of HDL and its component proteins and lipids within FF
together with the local expression of PON proteins within the
follicle. We are also currently investigating other antioxidant
enzymes, products of lipid peroxidation and antioxidant micro-
nutrients to provide a comprehensive evaluation of the role of
OS in the human ovarian follicle.
Beyond reproduction, the study of FF-HDL composition
may have implications in the pathogenesis of disease states
such as cardiovascular disease and diabetes. There is growing
evidence that modification of HDL structure and composition
has deleterious effects on its antioxidant and anti-inflammatory
capacity rendering it actually pro-oxidant, pro-inflammatory
and atherogenic (Navab, 2006; Ferretti et al., 2006).
Identification of the specific compositional alterations respon-
sible for these effects is difficult in systems containing LDL
due to its intricate interactions with HDL. Efforts to isolate
HDL by ultracentrifugation methods tend to distort its
structure and composition (Kunitake and Kane, 1982; Cabana
et al., 2003). The unique nature of FF possessing only HDL
may make it an ideal matrix to study the effects of specific
HDL alterations on its antioxidant and anti-inflammatory
We would like to acknowledge the assistance of Talia Mota in assist-
ing with embryo outcome data collection.
This work was not supported by any grant funding. Insti-
tutional, discretionary research funds available to Dr. Browne
and Dr. Fujimoto were used to support this work.
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Submitted on January 31, 2008; resubmitted on April 2, 2008; accepted on
April 16, 2008
Browne et al.